The long-term goal of the proposed research project is to gain a thorough understanding of the folding mechanisms of proteins by complementing the traditional bulk experimental methods with the novel approach of investigating the unfolding-refolding processes using the single molecule manipulation technique. The vast majority of proteins depend on the correct folding for their proper biological functions. It is also well established that incorrect folding of proteins can lead to aggregation and fibrillogenesis, which cause a number of human diseases, such as cystic fibrosis, prion and Alzheimer's diseases. A deeper understanding of the folding process will also greatly enhance our ability to design and fabricate new materials based on recombinant proteins, and synthesize novel enzymes with specific functions under unusual conditions. Therefore, the protein folding problem is not only a subject of intellectual curiosity, but also an area of enormous practical importance in medicine. Protein folding is a heterogeneous process in which a large number of microscopic pathways connect the unfolded conformations to the unique native structure. Ensemble measurements cannot fully address the problem of this heterogeneity. Alternative folding pathways can only be indirectly inferred, and certain functionally relevant regions of the energy landscape may not be accessible. Therefore, the single molecule technique, in which the protein molecules are studied one at a time, will have an important and complementary role to play in our search for a complete understanding of the protein folding problem. The focus of the project will be on the characterization of the energy landscape of three proteins with distinct folds. The specific aims of the project are: (1) Design and build a temperature controller for the AFM/single molecule manipulator to make temperature dependent measurements on mechanical unfolding of individual protein molecules. (2) Measure the roughness of the energy landscape of proteins in the temperature dependent mechanical unfolding experiments. Measurements will be carried out in both the constant force mode and the constant loading rate mode. (3) Characterize the transition states of three model proteins using the mechanical f-value analysis approach. Polymers of the wt and mutant proteins will be synthesized for these experiments. (4) Investigate the effects of molecular crowding on the unfolding-folding of proteins at the single molecule level. Different crowding agents will be used to elucidate the effects of crowding on the unfolding/refolding rates and refolding yield. [unreadable] [unreadable]